It is known to measure the concentration of an analyte such as a drug or hormone in a liquid by exposing the liquid to a receptor having binding sites on its molecule for the analyte, separating the receptor containing bound analyte from the liquid, measuring a value representative of the proportion of the available binding sites on the receptor molecule that have been occupied by analyte molecules (referred to as the fractional occupancy) and comparing that value with a corresponding measured value obtained with a solution of known concentration of the analyte.
The measurement of the value in question can be achieved by a back-titration technique involving contacting the receptor molecule containing bound analyte with a labelled version of the analyte. It is also possible to use, instead of labelled analyte, another labelled material able to occupy only those of the analyte binding sites on the receptor molecule that are not actually occupied by the analyte itself. These two systems are called competitive systems because the labelled analyte or other labelled material competes with the analyte being measured to occupy binding sites on the receptor molecule. In another alternative, the back-titration technique involves contacting the receptor molecule containing bound analyte with a material able to bind with the bound analyte or with only the binding sites occupied by bound analyte, this material being itself labelled or being subsequently labelled by attachment of a labelled marker. This system is known as a non-competitive system because there is no competition for binding sites.
In both the competitive and the non-competitive system the back-titration reagent (analyte or other material) is labelled with a marker. A variety of markers have been used, for example radioactive isotopes (radioimmunoassay), enzymes, chemiluminescent substances and fluorescent markers (fluoroimmunoassay), the latter being either a conventional fluorescent material such as fluorescein or a material which becomes fluorescent only on activation and estimation by time-resolved pulse fluorescence such as a europium or other lanthanide chelate, the magnitude of the fluorescence as revealed on scanning with a high-intensity light beam of appropriate wavelength being a measure of the amount of the labelled material taken up by the receptor molecule containing bound analyte.
Hitherto known assay techniques have depended either on a precise knowledge of the total amount of the receptor present in each sample or on the knowledge that the amount of the receptor remains precisely the same from sample to sample, especially from the unknown sample to the standard samples used for calibration purposes. They have also required an exact knowledge of the total sample volume. These requirements derive from the fact that the measured signal (e.g. fluorescence) in such systems is representative of the total amount of labelled material bound and, provided that not all the labelled material has been bound (in which event the system would be unresponsive to changes in the amount of analyte present), this total amount is dependent not only on the fractional occupancy of the binding sites on the receptor molecule, but also on the amount of receptor molecule present. In short, the fluorescent signal emitted in hithertoknown fluoroimmunoassay techniques has invariably depended in a complex (and, in practice, unknown) manner on the amount of receptor molecule used in the system and on the total amount of analyte present; this implies that both the amount of receptor and the sample volume used must be carefully standardised to ensure correct estimates of the analyte concentration in the test sample, such standardisation being a characteristic and essential feature of all hitherto-known fluoroimmunoassay techniques, particularly those described as competitive as defined above. It must be particularly emphasised that, in such techniques, the fractional occupancy of the antibody by analyte (and hence the fluorescent signal emitted) is itself dependent on the amount of receptor present; for example, increasing the number of receptor molecules by a given factor also increases the number of analyte molecules which it binds, but by a very different factor, causing the fractional occupancy of the receptor to markedly change. The amount of the labelled material binding to the receptor will also increase, but likewise in a non-proportional manner.
The necessity for standardisation of the amount of receptor used (a feature of hitherto-known fluoroimmunoassay techniques shared by analogous assay methods using other markers, such as radioisotopes) is not only experimentally demonstrable, but is theoretically predictable from consideration of the Mass Action Laws governing receptor/ligand interactions. Moreover, this requirement has also long been recognised as a serious disadvantage of these techniques, causing major problems in the quality control of immunoassay systems, particularly those in which the receptor (antibody) is attached to a solid support, and where it may be technically difficult to ensure that precisely the same amount of receptor is coupled to the solid material introduced into each sample incubation mixture. In this context, it must be noted that it has not been uncommon for those practised in the art to monitor or check the amount of receptor coupled to solid supports by labelling the receptor itself (e.g. with a radioisotope) to ensure constancy of the amount so coupled. Furthermore, it has also been recognised by those practised in the art that small variations in the amount of receptor coupled could be partially corrected for if, by use of such a receptor labelling technique, the deviation from the standard amount of receptor were known. Nevertheless, because the fractional occupancy by analyte of antibody binding sites depends in a complex and, in practice, unknown manner on both the amounts of receptor and analyte present, such approximate corrections are of limited usefulness and rarely if ever applied routinely.
For example, it has been proposed in International Patent Application WO 80/02076 to improve the quality control of immunoassays by providing two separate labels, preferably fluorescent labels, in the system, one label being attached to the competing ligand (as defined above), and a second label being attached to the receptor molecule. It is stated there that direct labelling of the receptor molecule has the advantage that the receptor molecule can be quantitatively detected during an immunoassay procedure independently of quantitative detection of the labelled (competing) ligand so that the immunoassay procedure may be made self-calibrating. In accordance with the preferred form of that invention, a fluorescent label on the receptor molecule and a fluorescent label on the labelled (competing) ligand are detected quantitatively while they are bound to each other and the quantity of the analyte present in an unknown sample is determined as a function of the ratio of the quantitative measurements of the two labels.
It is evident from the disclosures in WO/80/02076 that, apart from the use of a second label, the immunoassays described are conducted in a conventional manner. In so-called "competitive immunoassays", it is usual to employ sufficient receptor to bind 30-50% of both the labelled ligand and unlabelled analyte molecules present (at unlabelled analyte concentrations approaching zero). In so-called "non-competitive" assays, even higher concentrations of receptor are conventionally used, binding close to 100% of the analyte present. Clearly, in both these circumstances, the ratio of the signals emitted by the labelled receptor and labelled back-titration marker does not remain constant if the amount of receptor varies. (For example, in a non-competitive assay design, doubling the amount of the labelled receptor will not significantly increase the amount of analyte bound; hence the ratio will roughly halve. Similar considerations apply to the competitive systems discussed in WO/80/02076.)
The disclosures in WO 80/02076 thus merely provide a crude means of approximately correcting for minor variations in the amount of receptor present in assay systems of this type as described above, and are restricted in their application to this purpose. In short, the ratio of the quantitative measurements of the two labels (labelling receptor and competing ligand respectively) can be demonstrated, both theoretically and experimentally, not generally to constitute a measure of the amount of analyte in a conventional receptor-based assay system (such as an immunoassay), and the use of this ratio as a response variable will therefore generally give rise to analyte measurements which are grossly in error. Thus, though the disclosures in WO 80/02076 offer a technique (of limited usefulness) for simultaneously monitoring (and approximately correcting for) small variations in the amount of receptor present in individual sample incubation tubes as discussed above, they do not describe the design and operation of assay systems in which the measured ratio of two labels is an accurate and reliable measure of the analyte concentration in the sample.
Furthermore it must be emphasised that even when--as is usually the case--the amount of receptor can be very accurately standardised (i.e. the amount of receptor in every incubation tube can be held constant to very close limits and in which correction for the amount of receptor present as disclosed in WO 80/02076 is neither necessary nor useful), the resulting immunoassay system will normally require "calibration" (i.e. the inclusion of sets of assay standards containing known amounts of analyte) and this is conventional in the art. Insofar as the claim made in WO 80/02076 that labelling of the receptor renders an immunoassay `self-calibrating` possesses validity, it is only in the restricted sense that short-term variations in the efficiency of the signal detecting equipment (e.g. the radioisotope counter, fluorometer etc., depending on the label employed) can likewise be automatically corrected for (since a reduction in detection efficiency for one label would be expected to be accompanied by a roughly proportional reduction in detection efficiency for the other, causing the ratio to remain approximately constant).
Thus, in summary, the disclosure in WO 80/02076 provide at most the means of approximately correcting for a. small variations in the amount of receptor used in receptor-based assays and b. small, short term, variations in the signal detection efficiency of label-measuring equipment. These corrections are of questionable validity and restricted utility, and have not, for these reasons, been adopted in routine assay practice.